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An introduction is presented in which the editor discusses articles in the issue on topics including advancement in biological therapy; use of salivary gene transfer to treat preclinical animal models of serious systemic diseases; and the role of prostacyclin in pulmonary arterial hypertension.

Objectives Salivary glands are useful targets for gene therapeutics. After gene transfer into salivary glands, regulated secretory pathway proteins, such as human growth hormone, are secreted into saliva, whereas constitutive secretory pathway proteins, such as erythropoietin, are secreted into the bloodstream. Secretion of human growth hormone (hGH) into the saliva is not therapeutically useful. In this study, we attempted to redirect the secretion of transgenic hGH from the saliva to the serum by site-directed mutagenesis. Materials and Methods We tested hGH mutants first in vitro with AtT20 cells, a model endocrine cell line that exhibits polarized secretion of regulated secretory pathway proteins. Selected mutants were further studied in vivo using adenoviral-mediated gene transfer to rat submandibular glands. Results We identified two mutants with differences in secretion behavior compared to wild-type hGH. One mutant, ΔN1-6, was detected in the serum of transduced rats, demonstrating that expression of this mutant in the salivary gland resulted in its secretion through the constitutive secretory pathway. Conclusion This study demonstrates that mutagenesis of therapeutic proteins normally destined for the regulated secretory pathway may result in their secretion via the constitutive secretory pathway into the circulation for potential therapeutic benefit. [ABSTRACT FROM AUTHOR]

Oral Diseases (2011) , 476-483 Salivary glands are potentially a valuable target for gene therapeutics. Herein, we examined the expression and biochemical activity of human alpha-1-antitrypsin (hA1AT) produced in rodent submandibular glands after gene transfer. A serotype 5 adenoviral vector (Ad.hA1AT) was constructed and first characterized by dose response and time course studies using SMIE cells in vitro. hA1AT expression was analysed by ELISA and the biologic activity determined by the inhibition of human neutrophil elastase (hNE) and formation of hA1AT-hNE complexes. Ad.hA1AT was administered to submandibular glands of rats and mice. The levels and activity of hA1AT were analysed in saliva, serum and gland extracts. Treatment with endoglycosidase H and Peptide N-Glycosidase F was used to assess N-linked glycosylation. Transgenic hA1AT, expressed in submandibular glands following Ad.hA1AT administration, was secreted into the bloodstream, N-glycosylated and biochemically active. After in vivo gene transfer, rodent salivary glands can produce a non-hormonal, transgenic, secretory glycoprotein exhibiting complex and conformation-dependent biologic activity. [ABSTRACT FROM AUTHOR]

Objectives: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that has pro-apoptotic, pro-angiogenic and pro-inflammatory effects. In liver, TWEAK leads to proliferation of progenitor oval cells, but not of mature hepatocytes. This study evaluated the hypothesis that TWEAK overexpression in salivary glands would lead to the proliferation of a salivary progenitor cell. Methods: A recombinant, serotype 5 adenoviral vector encoding human TWEAK, AdhTWEAK, was constructed, initially tested in vitro, and then administered to male Balb/c mice via cannulation of Wharton’s duct. TWEAK expression in vivo was monitored as protein secreted into saliva and serum by enzyme-linked immunosorbent assays. Salivary cell proliferation was monitored by proliferating cell nuclear antigen staining and apoptosis was monitored using TUNEL staining. Results: AdhTWEAK administration led to a dose-dependent, transient TWEAK protein expression, detected primarily in saliva. Salivary epithelial cell proliferation was generalized, peaking on ∼days 2 and 3. TWEAK expression had no detectable effect on apoptosis of salivary epithelial cells. Conclusion: Transient overexpression of TWEAK in murine salivary glands leads to a general proliferation of epithelial cells vs a selective stimulation of a salivary progenitor cell. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Transforming growth factor beta (TGFbeta) plays a key role in the onset and resolution of autoimmune diseases and chronic inflammation. The aim of this study was to delineate the precise function of TGFbeta signaling in salivary gland inflammation. METHODS: We impaired TGFbeta signaling in mouse salivary glands by conditionally inactivating expression of TGFbeta receptor type I (TGFbetaRI), either by using mouse mammary tumor virus-Cre mice or by delivering adenoviral vector containing Cre to mouse salivary glands via retrograde infusion of the cannulated main excretory ducts of submandibular glands. RESULTS: TGFbetaRI-conditional knockout (TGFbetaRI-coko) mice were born normal; however, female TGFbetaRI-coko mice developed severe multifocal inflammation in salivary and mammary glands and in the heart. The inflammatory disorder affected normal growth and resulted in the death of the mice at ages 4-5 weeks. Interestingly, male TGFbetaRI-coko mice did not exhibit any signs of inflammation. The female TGFbetaRI-coko mice also showed an increase in Th1 proinflammatory cytokines in salivary glands and exhibited an up-regulation of peripheral T cells. In addition, these mice showed an atypical distribution of aquaporin 5 in their salivary glands, suggesting likely secretory impairment. Administration of an adenoviral vector encoding Cre recombinase into the salivary glands resulted in inflammatory foci only in the glands of female TGFbetaRI-loxP-flanked (floxed) mice (TGFbetaRI-f/f mice), but not in those of male and female wild-type mice or male TGFbetaRI-f/f mice. CONCLUSION: These results suggest that female mice are uniquely more susceptible to developing inflammatory disorders due to impaired TGFbeta signaling in their salivary glands. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To assess the safety and potential efficacy of etanercept in the treatment of Sjögren's syndrome (SS). METHODS: This pilot study was a 12-week randomized, double-blind, placebo-controlled trial of etanercept, with 14 subjects in each group. Patients received 25 mg of etanercept or placebo (vehicle) by twice-weekly subcutaneous injection. Patients met the American-European Consensus Group criteria for SS. The primary outcome required at least 20% improvement from baseline values for at least 2 of the following 3 domains: subjective or objective measures of dry mouth, subjective or objective measures of dry eyes, and IgG level or erythrocyte sedimentation rate (ESR). RESULTS: Of the 14 patients taking etanercept, 11 had primary SS and 3 had SS secondary to rheumatoid arthritis. Baseline measures did not differ between the 2 groups. Three etanercept-treated patients and 1 placebo-treated patient did not complete the trial. Five etanercept-treated patients and 3 placebo-treated patients showed improvement from baseline in the primary outcome variable at 12 weeks, but the difference was not statistically significant. There were no significant differences between the groups for changes in subjective measures of oral or ocular symptoms (by visual analog scale), the IgG level, Schirmer I test result, van Bijsterveld score, or salivary flow. At 12 weeks, the ESR had decreased in the etanercept group compared with baseline (P = 0.004); however, the mean reduction was only 18.6%. CONCLUSION: We found no evidence to suggest that treatment with etanercept at a dosage of 25 mg twice weekly for 12 weeks was clinically efficacious in SS. A larger trial will be necessary to definitively address the efficacy of etanercept in the treatment of SS. [ABSTRACT FROM AUTHOR]

Saliva is essential for the preservation of oral-pharyngeal health, and disorders of salivary physiology are associated with numerous oral and pharyngeal problems, particularly in older people. Although salivary function is remarkably intact in healthy aging, medical problems, medications, and head and neck radiotherapy can cause salivary dysfunction and complaints of xerostomia among older people. Sjogren's syndrome, an autoimmune exocrinopathy, is the most common medical disease associated with salivary dysfunction. Medications with anticholinergic side effects will impair salivary output, and head and neck radiotherapy for cancer will cause permanent destruction of salivary glands. Treatments for salivary problems are based upon establishing a diagnosis, protecting oral and pharyngeal health, stimulating remaining glands, and replacing lost salivary fluids. [ABSTRACT FROM AUTHOR]

Background: Recent investigations have demonstrated that parotid salivary dysfunction is not a normal process of aging, but may be the consequence of systemic conditions and their treatment, including medications and menopause. The purpose of this study was to assess longitudinally the influence of age, menopausal status, hormone replacement therapy, and other medications on stimulated parotid flow rates (SPFRs) in healthy women.Methods: Medical diagnoses, menopausal status, medication utilization, and 2% citric acid stimulated parotid salivas were collected from 396 women, aged 21 to 96 years, from the Baltimore Longitudinal Study of Aging (National Institute on Aging, National Institutes of Health) over a 17-year span by three investigators.Results: There was no overall longitudinal effect of time on SPFR. Age at first visit was a significant predictor of a decrease in SPFR when adjusted for time and xerostomic medications. However, the deleterious effect of taking one xerostomic medication was equivalent to approximately 14 years of aging. Menopausal status and hormone replacement therapy were not consistently associated with diminished SPFR.Conclusions: These results suggest that menopause and hormone replacement therapy are not associated with parotid salivary dysfunction. Aging may have a statistically significant yet small deleterious influence on SPFR; however, the adverse influence of xerostomic medications is much larger. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate if two pharmacological agents, Tempol and D-methionine (D-met), are able to prevent oral mucositis in mice after exposure to ionizing radiation ± cisplatin.Methods and Materials: Female C3H mice, ∼8 weeks old, were irradiated with five fractionated doses ± cisplatin to induce oral mucositis (lingual ulcers). Just before irradiation and chemotherapy, mice were treated, either alone or in combination, with different doses of Tempol (by intraperitoneal [ip] injection or topically, as an oral gel) and D-met (by gavage). Thereafter, mice were sacrificed and tongues were harvested and stained with a solution of Toluidine Blue. Ulcer size and tongue epithelial thickness were measured.Results: Significant lingual ulcers resulted from 5 × 8 Gy radiation fractions, which were enhanced with cisplatin treatment. D-met provided stereospecific partial protection from lingual ulceration after radiation. Tempol, via both routes of administration, provided nearly complete protection from lingual ulceration. D-met plus a suboptimal ip dose of Tempol also provided complete protection.Conclusions: Two fairly simple pharmacological treatments were able to markedly reduce chemoradiation-induced oral mucositis in mice. This proof of concept study suggests that Tempol, alone or in combination with D-met, may be a useful and convenient way to prevent the severe oral mucositis that results from head-and-neck cancer therapy. [ABSTRACT FROM AUTHOR]

Marie Ussing Nylen was a trail blazing scientist and administrative leader at the US National Institutes of Health. She accomplished this when it was extremely difficult for a woman to do so. She was also a whole person - a wife, mother, and talented athlete, that is, a well-rounded person by any definition. She was a gift to dental and oral science, as well as to those fortunate enough to know and work with her., (Published 2022. This article is a U.S. Government work and is in the public domain in the USA.)

We evaluated late effects of AdhAQP1 administration in five subjects in a clinical trial for radiation-induced salivary hypofunction (http://www.clinicaltrials.gov/ct/show/NCT00372320?order=). All were identified as initially responding to human aquaporin-1 (hAQP1) gene transfer. They were followed for 3-4 years after AdhAQP1 delivery to one parotid gland. At intervals we examined salivary flow, xerostomic symptoms, saliva composition, vector presence and efficacy in the targeted gland, clinical laboratory data and adverse events. All displayed marked increases (71-500% above baseline) in parotid flow 3-4.7 years after treatment, with improved symptoms for ~2-3 years. There were some changes in [Na + ] and [Cl - ] consistent with elevated salivary flow, but no uniform changes in secretion of key parotid proteins. There were no clinically significant adverse events, nor consistent negative changes in laboratory parameters. One subject underwent a core needle biopsy of the targeted parotid gland 3.1 years post treatment and displayed evidence of hAQP1 protein in acinar, but not duct, cell membranes. All subjects responding to hAQP1 gene transfer initially had benefits for much longer times. First-generation adenoviral vectors typically yield transit effects, but these data show beneficial effects can continue years after parotid gland delivery.

For many years, our research group worked to develop gene transfer approaches for salivary gland disorders that lacked effective conventional therapy. The purpose of this chapter is to describe and update key methods used in this process. As described in our original chapter from the 2010 volume, we focus on one clinical condition, irradiation-induced salivary hypofunction, and address the choice of transgene and vector to be used, the construction of recombinant viral vectors, how vector delivery is accomplished, and methods for assessing vector function in vitro and in an appropriate animal model.

Purpose: Clinician-scientists are considered an endangered species for many reasons, including challenges with establishing and maintaining a career pipeline. Career outcomes from yearlong medical and dental students' research enrichment programs have not been well determined. Therefore, the authors assessed career and research outcome data from a cohort of participants in the National Institutes of Health (NIH) Clinical Research Training Program (CRTP)., Method: The CRTP provided a yearlong mentored clinical or translational research opportunity for 340 medical and dental students. Of these, 135 completed their training, including fellowships, from 1997 to January 2014. Data for 130 of 135 were analyzed: time conducting research, types of public funding (NIH grants), and publications from self-reported surveys that were verified via the NIH Research Portfolio Online Reporting Tools Web site and PubMed., Results: Nearly two-thirds (84 of 130) indicated that they were conducting research, and over half of the 84 (approximately one-third of the total cohort) spent more than 25% of time conducting research. Of those 84, over 25% received grant support from the NIH, and those further in their careers published more scholarly manuscripts., Conclusions: Data suggest that the CRTP helped foster the careers of research-oriented medical and dental students as measured by time conducting research, successful competition for federal funding, and the publication of their research. Longer follow-up is warranted to assess the impact of these mentored research experiences. Investments in mentored research programs for health professional students are invaluable to support the dwindling pipeline of biomedical researchers and clinician-scientists.

Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands functionally compromised post IR.